Altering viral tropism

ABSTRACT

Methods of altering retroviral tropism have been discovered. Such methods are useful, e.g., for developing retroviral vectors for gene therapy.

TECHNICAL FIELD

This invention relates to virology.

BACKGROUND

Recombinant retroviral vectors are attractive vehicles for gene deliverybut they generally lack the cell specificity that is desirable forapplications involving gene therapy. For example, the Murine LeukemiaVirus (MLV) ecotropic envelope protein (Moloney MLV envelope protein;MoMLV envelope protein) binds to an amino acid transporter that isexpressed only in mouse cells and the cells of closely related species(Albritton et al., 1989, Cell 57:659-666), but not in human cells. Hostrange is determined by regions of variable sequences (termed VRA, VRB)within the extracellular domain (SU) of envelope protein (envelope).

SUMMARY

The invention is based in part on the discovery that retroviral tropismof ecotropic MLV can be altered or redirected using heterologous shortpeptide ligands inserted within the retroviral envelope protein of thisvirus to form chimeric envelope proteins. Such chimeric envelopeproteins can be incorporated into a viral vector to create a pseudotypedvirus. Wild-type envelope sequence does not have to be deleted for thechimeric envelope proteins to be effective for binding or transductionof a pseudotyped virus that incorporates them nor do they require thepresence of an intact wild-type envelope for efficient transduction. Inaddition, it has been discovered that the length and position of theinserted peptide ligand can affect viral tropism. Thus, the inventionrelates to a novel method for targeting retroviruses to specific cellsby modifying viral envelope proteins. The chimeric envelope proteins areuseful, e.g., for creating a vector that can transduce a target cell(for example, a human cell) and for introducing a gene into such atargeted cell, for example, to selectively target and destroy humancancer cells.

In one embodiment, the invention is a recombinant chimeric envelopeprotein that includes a wild-type ecotropic Murine Leukemia Virus (MLV)envelope protein and a heterologous short peptide ligand inserted withinthe MLV envelope protein. The invention also includes a nucleic acidsequence encoding such recombinant envelope proteins and plasmid vectorsthat contain such sequences. The heterologous short peptide ligand canbe an RGD ligand, a human epidermal growth factor receptor (HRG) ligand,or a gastrin releasing protein (GRP) ligand. In some aspects of theinvention, the heterologous short peptide ligand is flanked by at leastone cysteine on each side. In another aspect of the invention, theheterologous short peptide ligand is inserted into a conserved region ofa wild-type envelope protein.

The invention also includes a vector comprising a nucleic acid or geneencoding a chimeric envelope protein that contains a heterologous shortpeptide ligand. The vector can also contain a nucleic acid sequence thatcodes for a therapeutically useful protein.

In another embodiment, the invention is a recombinant retroviralparticle that contains a chimeric envelope protein containing aheterologous short peptide ligand. In some embodiments, such recombinantretroviral particles can infect a mouse cell or a target host cell. Inother embodiments, the recombinant retroviral particle cannot infect amouse cell.

In another aspect, the invention includes a method of altering murineleukemia virus (MLV) retroviral tropism by introducing into the genomeof an MLV a nucleic acid sequence that codes for a recombinant envelopeprotein that codes for a heterologous short peptide ligand. In someembodiments of the invention, the virus cannot express wild-typeenvelope protein. In another embodiment, the heterologous short peptideligand is inserted into a conserved region of a wild-type envelopeprotein.

The invention also includes a method of identifying a chimeric envelopeprotein that alters viral tropism by introducing into the genome of anMLV a nucleic acid sequence encoding a recombinant envelope proteincontaining a heterologous short peptide ligand thus making a recombinantvirus, infecting a target host cell with the virus, and assayingtransduction of the target host cell by the virus, such thattransduction of the host cell by the virus indicates that therecombinant envelope protein alters viral tropism. In this method, theheterologous short peptide ligand can be located in a conserved regionof the MLV envelope protein, and the target host cell can be a humancell. More specifically, the target host cell can be a cancer cell or acell that contains a defective gene. In some embodiments, the chimericenvelope protein contains an RGD ligand, an HRG ligand, or a GRP ligand.

In another aspect, the invention includes a method of delivering a geneto a cell by infecting a cell with a virus, e.g., a retrovirus,containing a chimeric envelope protein comprising a heterologous shortpeptide ligand and a gene. The ligand can be an RGD ligand, an HRGligand, or a GRP ligand. The host cell can be an animal cell, e.g., amammalian or human cell, e.g., a cancer cell. Further, the cell can bein an animal, e.g., in a human.

The invention also includes a method of treating cancer by infecting acancer cell with a virus, e.g., a retrovirus, containing a chimericenvelope protein that includes a heterologous short peptide ligand and agene that can be used to treat the cancer. The cancer to be treated canbe in an animal, such as a mammal, e.g., a human subject. In someaspects, the therapeutically useful gene codes for thymidine kinase.

A “heterologous short peptide ligand” is a peptide between 3 and 90,e.g., 3 and 83, or 6 and 21 amino acids in length, that can specificallybind to a receptor on a cell. The short peptide sequence is heterologouswith respect to the wild-type envelope protein into which it isinserted. Examples of heterologous short peptide ligands include RGDligands, GRP, and HRG ligands as described herein. Other heterologousshort peptide ligands can be identified using methods known in the artand the methods described herein.

A “chimeric envelope protein” is a polypeptide containing a retroviralwild-type envelope protein sequence (e.g., an ecotropic MLV envelopeprotein) into which has been inserted a heterologous short peptideligand. The chimeric envelope protein may contain the complete sequenceof the envelope protein from which it is derived. In some cases aportion (e.g., 1 to about 110 amino acids) of the wild type envelopeprotein is deleted. A nucleic acid sequence coding for a chimericenvelope protein contains a nucleic acid sequence coding for an envelopeprotein and a nucleic acid sequence coding for a heterologous shortpeptide ligand that is inserted in-frame.

A “target host cell” is a cell that can be transduced by a pseudotypedvirus containing a chimeric envelope protein. In general, a target hostcell is not from the same species as the host cell for the wild-typevirus from which the pseudotyped virus is derived. Typically, thepseudotyped virus will bind only to the target host cell and not toother cell types. If the parent virus (i.e., the wild-type virus) usedto produce the pseudotyped virus can bind to cells of the host, it isgenerally desirable to reduce or eliminate this binding, for example, bymutation of the binding site. Host cells can be mammalian, e.g., dog,cat, cow, horse, monkey, or human cells. A host cell can be isolatedfrom a host animal and cultured, or cultured and reintroduced into thehost. Alternatively, a host cell can be within the host animal, e.g., ina specific tissue in the host such as muscle, blood progenitor or matureblood cell, liver, kidney, or a tumor or other diseased tissue.

A “therapeutically useful gene” is a gene encoding a nucleic acid orpolypeptide that, when expressed in a cell, for example, a target hostcell, can provide a therapeutic effect.

A molecule that specifically binds to a second molecule (e.g., to aparticular receptor on a cell) is a molecule that the second molecule,but does not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the second molecule.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph illustrating the results of an experiment in whichNIH 3T3 cells and A375 human melanoma cells were transduced by RGD₁₃viruses.

FIG. 2 is a bar graph showing the results of experiments testing theability of RGD₂₁ viruses to transduce NIH 3T3 cells and A375 humanmelanoma cells.

FIGS. 3A-3B are bar graphs illustrating transduction experiments testingthe requirement of the RGD sequence for transduction of human cells. (A)Transduction of NIH 3T3 infected with an RGD₂₁ or RGE₂₁ virus, and (B)Transduction of A375 human melanoma cells infected with an RGD₂₁ orRGE₂₁ virus.

FIGS. 4A-4B are bar graphs showing the results of experiments testingthe effect of pretreatment with antibodies to integrin receptors ontransduction of human cells by RGD viruses (A) NIH 3T3 cells; (B) A375human melanoma cells.

FIG. 5 is a bar graph showing the results of experiments testing theability of GRP viruses to transduce human cells.

FIGS. 6A-6C are bar graphs showing the results of experiments examiningthe requirement of the GRP receptor for transduction of human cells byGRP viruses. (A) Antibodies to GRP block transduction of human cells byGRP viruses. (B) Requirement of the GRP receptor for transduction ofhuman 293 cells. (C) Requirement of the GRP receptor for transduction ofmouse cells by GRP-2, GRP-3 and GRP-5 viruses.

FIGS. 7A-7B are bar graphs showing the results of experiments testingthe ability of HRG viruses to transduce NIH 3T3 cells and MDA-MB-453breast carcinoma cells. (A) Transduction of NIH 3T3 cells by HGRviruses. (B) Transduction by HRG-1 or HRG-8 virus after pretreatment ofNIH 3T3 and MDA-MB-453 breast carcinoma cells with antibodies to HER-3and HER-4 receptors.

FIG. 8 is a representation of the nucleic acid sequence of MoMLVenvelope protein (SEQ ID NO:4).

DETAILED DESCRIPTION

The invention provides a strategy for altering the host range ofecotropic retrovirus vectors using a recombinant envelope protein thatcontains a heterologous short peptide ligand (chimeric envelopeproteins). Viruses expressing such chimeric envelope proteins(pseudotyped virus) can transduce human cells without removal of theN-terminal region of the naturally occurring envelope protein orco-expression of wild-type envelope protein. Furthermore, it is notnecessary to delete portions of the wild-type envelope protein sequenceto obtain a chimeric envelope protein that, when present in apseudotyped virus, can alter host specificity and infect with reasonableefficiency. Depending on the site in the envelope protein of insertionof the heterologous short peptide ligand, the pseudotyped viruscontaining the resulting chimeric envelope protein can transduce onlytarget host cells. Target host cells can be any eukaryotic cell typeexpressing a sequence on the cell surface that can bind to theheterologous short peptide ligand. In general, a target host cell is amammalian cell, e.g., a human cell. In one embodiment, a heterologousshort peptide ligand is inserted into an extracellular portion of an MLVenvelope protein. For example, the heterologous short peptide ligand canbe inserted into a conserved region of the envelope protein, or into avariable region.

Heterologous Short Peptide Ligands

Heterologous short peptide ligands for use in the invention can be thosealready identified in the art. Many peptide sequences that bind to cellsurface proteins have been identified. Some such sequences are so-called“designer” peptides whose affinity for receptors surpasses that ofwild-type peptide sequences. One example of such a designer peptide isthe heregulin peptide described in Table 2 and Example 8. Additionalexamples of cell surface proteins/receptors that bind to ligands areinclude flt-3 receptor/flt3 ligand (FL), transferrinreceptor/transferrin, erythropoietin receptor/erythropoietin (EPO)peptides (e.g., the consensus sequence IEGPTLRQWLAARA; SEQ ID NO:1;Cwirla, et al., 1997, Science 276:1696-1699), CD34/variable sequence ofa binding antibody; c-kit/stem cell factor (binding region peptide);human melanoma-associated chondroitin sulfate proteoglycan(MCSP)/anti-MCSP antibody (used for the detection of antibodies); MHCclass I/Semiliki Forest Virus binding sequence; MHC class II low densitylipoprotein receptor/variable sequence of antibody; mucins (surfaceglycoproteins overexpressed in numerous cancers)/binding peptidesequence (APDTP; SEQ ID NO:2); IL-2 receptor/IL-2; surface glycoproteinhigh-molecular-weight melanoma-associated antigen (HMW-MAA)/bindingregion from variable sequence of antibody.

Heterologous short peptide ligands suitable for use in the invention canalso be identified using methods known in the art. Such methods includescreening phage selected for binding to the extracellular domain of acell surface protein (i.e., a cell surface protein expressed on a hosttarget cell). Nucleic acid sequences coding for such peptides are thencloned into wild-type envelope protein to produce chimeric envelopeproteins. In another method using phage library, targeting to variousorgans can be achieved by injecting a phage display library into animalsand identifying the peptides localized in each organ. This method hasbeen successfully used to identify short peptides targeted to, e.g.,kidney cells (CLPVASC, SEQ ID NO:3; and CGAREMC, SEQ ID NO:5) and tobrain cells (CLSSRLDAC, SEQ ID NO:6; WRCVLREGPAGGCAWFNRHRL; SEQ ID NO:7)(Pasqualini et al., 1996, Nature 380:364-366). Similarly, recombinantpeptide libraries can also be screened for peptides that specificallybind to a protein that is expressed on a target host cell (Pasqualinisupra; Wrighton et al., 1996, Science 273:458-464; Cwirla et al., 1997,Science 276:1696-1699; Arap et al., 1998, Science 279:377-380).

Chimeric Envelope Proteins and Libraries

Envelope proteins are known in the art. In particular, the ecotropicmurine leukemia virus protein has been extensively studied. The sequenceof the MoMLV envelope protein (gp70) is shown in FIG. 8. The sequencecoding for the extracellular domain (SU) region of the envelope proteinextends from nucleotides 5612-6919. The transmembrane region andcytoplasmic tail extend from nucleotides 6920-7507. There is a signalpeptide sequence at the beginning of the SU, that localizes the proteinto the cell membrane. Clones containing MoMLV envelope protein arecommercially available (e.g., Stratagene, La Jolla, Calif.).Heterologous short peptide ligands are inserted in the extracellulardomain of the envelope protein. In general, chimeric envelope proteinscontaining insertions near the N-terminus and in the proline-rich region(PRR region) of the envelope protein are less effective for alteringviral tropism than insertions at other positions within the protein.Examples of specific insertion locations that are effective aredescribed herein, and in detail in the Examples.

Transduction efficiency also depends on the presentation of the ligandwithin the envelope. In some embodiments of the invention, cysteineresidues flank the inserted heterologous short peptide ligand. Suchresidues are expected to form a disulfide bond that facilitates ligandpresentation. Cysteines that flank the heterologous short peptide ligandcan be immediately adjacent to the short peptide sequence. In someembodiments of the invention, such sequences are 2, 3, 4, 5, or about10, 20, 30, 50, or 100 amino acid residues from the ends of theheterologous short peptide ligand. The cysteines can be added to theenvelope protein that is being engineered to contain the heterologousshort peptide ligand, or the heterologous short peptide ligand can bepositioned so that one or two cysteines that naturally occur in thewild-type protein are flanking cysteines.

The invention includes the generation and screening of chimeric envelopeprotein libraries. In one method of generating such libraries, a clonedenvelope protein (e.g., a cloned MoMLV envelope protein) or a portion ofan envelope protein, generally the sequence coding for the extracellulardomain of the envelope protein, is cut with restriction enzyme.Typically, the restriction enzyme(s) are four-base cutters and thereaction is carried out in the presence of ethidium bromide. Thepresence of ethidium bromide limits the number of times a plasmid willbe cut by the restriction enzyme, typically to one cleavage per plasmid,thus resulting in linearized plasmid. The ends of the plasmid aretreated to produce blunt ends. A blunt-ended nucleic acid sequenceencoding the heterologous short peptide ligand of interest is preparedand ligated into the linearized plasmid preparation. Differentrestriction enzymes can be used to increase the number of sites intowhich sequences coding for the heterologous short peptide ligand can beinserted. The plasmids can then be transfected into bacteria. Plasmidsare examined for heterologous short peptide ligand sequence and thelocation of the heterologous short peptide ligand within the envelopesequence using methods known in the art, e.g., PCR and Southern blotanalysis. If a portion of the envelope sequence was used forconstruction of a sequence containing the inserts of heterologous shortpeptide ligand, then the portion of the envelope sequence containing theheterologous short peptide ligand is cloned into a plasmid containingenvelope sequence to generate a sequence coding for a complete envelopeprotein containing a heterologous short peptide ligand (i.e., a chimericenvelope protein).

Pseudotyped Viruses

To produce pseudotyped viruses containing a specific chimeric envelopeprotein, a plasmid that contains a sequence that codes for the chimericenvelope protein is co-transfected into a packaging cell with apackaging construct, e.g., the packaging cell line, Anjou 65 (Pear etal., 1993, Proc. Natl. Acad. Sci. USA 90:8392-8396) and the packagingconstruct LGRNL (Yee et al., 1994, Methods Cell Biol. 43:99-112). Theresulting cell is maintained under conditions such that virus isproduced. The resulting pseudotyped virus can then be tested for itsability to transduce a natural host cell (e.g., a murine cell when thepseudotyped virus is derived from an ecotropic virus) and a target hostcell (e.g., a human cell). A virus may be able to transduce target hostcells from more than one species, depending on the ability of theheterologous short peptide ligand to bind to the corresponding receptorsexpressed on cells from various species. A pseudotyped virus may alsotransduce more than one cell type, e.g., those cell types that expressthe targeted receptor.

Cells can be tested for transduction using methods known in the art. Forexample, Southern blotting can be used to test for insertion ofretroviral sequence into a host cell genome. The pseudotyped virus mayinclude a selectable gene, e.g., a gene that confers drug resistancesuch as neo. In this case, an infected host cell is incubated in thepresence of the drug. Cells that have been successfully transducedsurvive in the presence of the drug. Pseudotyped virus can also betested for the efficiency of transduction. In general, pseudotypedviruses with the greatest efficiency of transduction of host cells arepreferred, e.g., for delivery of a gene to a cell as in gene therapy.

In some cases, it is desirable to introduce an additional gene, e.g., atherapeutically useful gene, into the pseudotyped virus and/or into thepackaging cell. Such a gene can be either on the packaging construct oron a separate plasmid. Therapeutically useful genes include those thatreplace or supplement the product of a defective gene in the target hostcell. Examples of such genes include the globin genes delivered to bonemarrow progenitor cells to treat sickle cell anemia or a thalassemia,and factor VIII or factor IX genes delivered to blood progenitor cellsto treat hemophilia. Also included are genes that encode proteins,antisense transcripts, or ribozymes that can be delivered to cells thatexpress CD4 and can be used to treat HIV, genes that encode therapeuticantibodies, growth factors, or cytokines to be expressed by host targetcells. Therapeutically useful genes also include genes that can be usedfor cancer therapy such as genes that code for proteins that destroy thetarget host cell (either directly or after treatment of such cells witha drug) and genes that code for antisense transcripts or ribozymes thatinterfere with target host cell function.

Gene Delivery

Pseudotyped viruses as described herein are useful as vectors fordelivery of genes to cells, e.g., for ex vivo or in vivo gene therapy.In addition to the advantages conferred by using a retrovirus (e.g.,integration of the transferred gene(s)) an advantage of the pseudotypedviruses is that they can be designed to transduce specific cell types.For example, as discussed supra, some cancer cells overexpress specificcell surface proteins. Such proteins can be used as the target receptorsfor a heterologous short peptide ligand in the chimeric envelopeprotein, thus conferring specificity on a pseudotped virus thatexpresses the chimeric envelope protein. A further advantage of using anenvelope protein from a murine ecotropic virus for making the chimericenvelope proteins, is that the naturally occurring envelope protein willtarget only murine cells. Thus, the pseudotyped virus, if used to infecta non-murine cell such as a human cell, will transduce only those cellsexpressing the receptor for the heterologous short peptide ligand. Suchpseudotyped viruses whose tropism has been altered can also be selectedfor the inability to transduce a murine cell and the ability totransduce a cell expressing a target receptor. As described herein, itis also possible to make and identify, depending on the location of theheterologous short peptide ligand within the chimeric envelope protein,pseudotyped virus that can only transduce a target host cell, i.e.,cannot transduce a murine cell.

Pseudotyped viruses made using the methods described herein can be usedto introduce a gene into an animal or into cells of an animal that arecultured in vitro then reintroduced into the animal (ex vivo genetherapy). In addition, a pseudotyped virus that contains atherapeutically useful gene can be introduced into an animal model for adisease such as cancer. Therapeutically useful genes are discussed supraand include genes that code for a protein that is defective in theanimal or for a gene that provides a novel property to a cell, forexample, drug sensitivity to a tumor cell. The pseudotyped virus may beintroduced using any method known in the art. For example, thepseudotyped virus can be introduced locally (for example, near a tumor)or systemically. In the latter case, it may be desirable toimmunosuppress the animal using methods known in the art to minimize theimmune response to the pseudotyped virus.

The use of retroviral vectors is known in the art and the pseudotypedviruses described herein provide advantages over the presently usedvectors. In particular, the target cell specificity and the limitedability of the pseudotyped vectors to replicate in target host cells arean improvement over those systems in which the viral vector infectscells other than those where gene delivery is desired and in which viralreplication may interfere with the cellular metabolism.

Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470), or by stereotactic injection (see e.g., Chen et al., 1994,Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is embedded. Alternatively, where the completegene delivery vector (e.g., a pseudotyped virus) can be produced intactfrom recombinant cells, the pharmaceutical preparation can include oneor more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Viruses

In general, the invention uses envelope proteins derived from ecotropicretroviruses such as the Moloney murine leukemia virus (MoMLV). Alsouseful in the invention are viruses that express a glycoproteinenvelope. Such viruses include the murine leukemia virus family (MLV)(e.g., amphotropic, ecotropic, and xenotropic viruses). Amphotropicviruses can typically infect human cells, whereas ecotropic viruses caninfect only host cells of the species in which they originated. Thus,murine ecotropic viruses cannot naturally infect human cells. Hosttargeting (tropism) of viruses other than retroviruses can also bemodified using envelope proteins. Examples of such viruses includeadenovirus (by inserting a heterologous short peptide ligand into thefiber of the surface protein of adenovirus) and vesicular stomatitisvirus (VSV-G), which is an attractive candidate as the pseudotyped viruscan be concentrated by high speed centrifugation without significantloss of titre. Cells (e.g., human) can be targeted using pseudotypedviruses derived from many different viruses, including those that thatenter the cell through an endocytic process (e.g., Moloney MLV, asdescribed herein), or by a virus that fuses at the cell surface (as withamphotropic MLV). Additional examples of viruses whose targeting can bemodified using the methods described herein include gibbon ape leukemiavirus, influenza virus (chimeric hemagglutinin), spleen necrosis virus,reticuloendotheliosis virus strain A (REV-A), herpes virus (HSV-1),human immunodeficiency virus (HIV; Naldini et al., 1996, Science 272:263-267), and various species of hepatitis virus.

In general, a library of chimeric envelope proteins containingheterologous short peptide ligands that are useful for altering hostrange of a virus are made as described herein. The chimeric envelopeproteins can first be screened for their ability to bind to the receptorto which the heterologous short peptide ligand binds when it is notinserted into an envelope protein. The chimeric envelope proteins areincorporated into a virus (thus making a pseudotyped virus) and testedfor their ability to specifically transduce a target host cell.

Uses of Pseudotyped Viruses

It is demonstrated herein that chimeric envelope proteins enabletransduction of human cells by a pseudotyped virus derived from an MLV.In addition, transduction of human cells with pseudotyped MLV does notoccur with heterologous short peptide ligand insertions (e.g., RGDpeptide ligands) in the PRR (proline-rich region) or C-terminal regionof the envelope, although pseudotyped viruses containing such insertionscan transduce mouse cells. Some viruses bearing insertions (e.g., of RGDpeptide ligands) at the N-terminus or VRA region (RGD₁₃-4,5,8 andRGD₂₁-2) transduce human but not mouse cells. Thus, the position of theinserted ligand can dictate tropism.

Transduction efficiencies differ between different RGD pseudotypedviruses, indicating that the precise location of the ligand withinenvelope is important. In one aspect, the invention includes methods foroptimizing the location of heterologous short peptide ligands (e.g., RGDpeptide ligands) within the envelope protein. In general, RGD₁₃ andRGD₂₁ ligands transduce NIH 3T3 cells with comparable efficiencies.Thus, the envelope protein can accommodate ligands of different sizesand remain effective for transduction. Longer ligands can be moredisruptive to the structure of the envelope protein, but may also haveincreased affinity for the target receptor. Such ligands can includerepeats of the heterologous short peptide ligand sequence (for example,2 copies, three copies, 4 copies, five copies, or up to ten copies).

The methods described herein for altering the tropism of a retroviruscan be used to selectively target and destroy human cancer cells. Forexample, many cancer cells overexpress specific cell surface receptors.As discussed below, Moloney murine leukemia virus (MLV) envelopeproteins bearing heterologous short peptide ligands for gastrinreleasing protein (GRP) and human epidermal growth factor receptors(HRG) were generated. More than twenty MLV chimeric envelope proteinsthat contain the GRP or a modified HRG peptide ligand were inserted atvarious locations within envelope. Pseudotyped viruses containing thesechimeric envelope proteins selectively transduce human cancer cell linesthat overexpress the cognate receptor. For both GRP and HRG viruses,some insertions within the N-terminal region or VRA (a variable region)of the envelope protein interfere with transduction of mouse cells.Several of these GRP viruses transduce cells expressing the GRP receptorindicating that tropism is altered. Thus, for production of selectivetargeting retroviral vectors, the N-terminal region and VRA can be theoptimal locations for ligand insertion. Transduction by virusescontaining the larger HRG ligand is, in general, decreased relative totheir GRP counterparts and several HRG viruses are unable to transducemouse or human cells. However, the HRG ligands used in these experimentsare approximately twice as long as the GRP ligands. This furtherdemonstrates that short ligands are generally more efficient for use inthe methods of the invention and are an improvement over those generallyused previously.

The new methods include using a pseudotyped virus containing a chimericenvelope protein to deliver a therapeutically useful gene to a cell.This was demonstrated by showing that pseudotyped virus targeting theGRP receptor can deliver the thymidine kinase (TK) gene to humanmelanoma and breast cancer cells, which makes these transgenic cellssusceptible to the antiviral agent, ganciclovir. Furthermore, thetransduced cells were killed by the subsequent addition of ganciclovir,demonstrating that heterologous short peptide ligands inserted atappropriate locations in an ecotropic envelope protein (e.g., MoMLVenvelope protein) can selectively target a retrovirus to a human cancercell and deliver a therapeutically useful gene. These experiments alsodemonstrate the utility of the method and constructs to selectivelytarget cancer cells overexpressing GRP or HRG receptors and deliver atherapeutically useful gene. The method can also be used, e.g., tointroduce a gene or other nucleic acid sequence into any cell type thatexpresses a receptor that can be targeted as described herein. Thisincludes introducing a gene or other nucleic acid into a cell in cultureor in an animal (e.g., a non-human mammal such as a mouse, rat, sheep,cow, or goat). For example, in a mixed culture of cells, the method canbe used to deliver a gene to a single cell type in the culture, e.g., toprovide a marker for the cell type or to introduce a drug-resistancegene to that cell type.

A pseudotyped virus containing a chimeric envelope protein can begenerally useful in gene therapy methods for animals and humans. Genetherapy strategies have been proposed for many human diseases, includingrare heritable genetic defects, of which there are more than 4000, andmany common diseases including cancer, AIDS, hypertension, and diabetes(Anderson, 1992, Science 256: 808-813; Friedmann, 1992, Nature Genet.2:93-98; Russell, 1993, Cancer J. 6:21-25). The invention therefore hasan important application in many areas of human medicine.

EXAMPLES Example 1 Cell Lines

In experiments described herein, Anjou 65 (Pasqualini and Ruoslahti,1996, Nature 380:364-366), NIH 3T3, XC cells (Wrighton et al., 1996,Science 273:458-464), A375 human melanoma, HT 1080 human fibrosarcoma,and MDCK canine kidney cells were each cultured separately as monolayersin Dulbecco's modified Eagle medium (DMEM; Gibco BRL) supplemented with10% fetal bovine serum (Hyclone), 2 mM glutamine, and 5 mM HEPES. Allcell lines, except for Anjou 65, were obtained from the American TypeCulture Collection (ATCC) and maintained at 37° C. in a 5% CO₂atmosphere.

Example 2 Construction of Short Peptide RGD Ligand Viruses

To test for the ability of heterologous short peptide ligands toredirect the host range of a virus, more than 40 chimeric envelopeproteins containing in-frame insertions of either a 13 or 21 amino acidRGD peptide (RGD₁₃ or RGD₂₁, respectively; Table 1) were examined. Thesequences of the RGD₁₃, RGD₂₁, and RGE₂₁ ligands are shown in Table 1.For the chimeric envelope proteins RGD₁₃ 1-26, RGD₂₁ 1-16 and RGE₂₁ 1-5,the position of ligand insertion, number of inserts, and any additionalmodifications are indicated in Table 1.

The heterologous short peptide ligands were introduced into envelopeprotein to form chimeric envelope proteins. To construct the chimericenvelope proteins, the extracellular domain (gp70) of ecotropic MLVenvelope gene was linearized at random locations by partial digestionwith blunt-end restriction endonucleases in the presence of 50 to 400ng/ml ethidium bromide. The 13 amino acid RGD sequence (CAAAGRGDSPTRC;RGD₁₃; SEQ ID NO:8) was derived by annealing two oligonucleotides,RGD₁₃-A (TGCGCGGCCGCTGGCCGTGGCG-ATTCTCCCACGCGTTGT; SEQ ID NO:9) andRGD₁₃-B (ACAACGCGTGGGAGAATCGCC-ACGGCCAGCGGCCGCGCA; SEQ ID NO:10). Theannealed sequence was ligated into the linearized envelope plasmid andsubclones screened for insert position and orientation using standardtechniques. The resultant chimeric envelope proteins were cloned intothe envelope expression vector, pCEE (MacKrell et al., 1996, J. Virol.70:1768-1774). The RGD₁₃-3 chimeric envelope proteins were constructedby insertion of a Nae I linker at the C-terminus of the signal sequenceof wild type envelope and the annealed RGD oligonucleotides were clonedinto the Nae I site. Chimeric envelope proteins with the 21 amino acidRGD sequence CAAAQGATFALRGDNPQGTRC; RGD₂₁; SEQ ID NO:11) wereconstructed by restriction endonuclease digestion of RGD₁₃ envelopeswith Not I and Mlu I and insertion of the RGD₂₁ annealedoligonucleotidesRGD21-A(GGCCGCTCAAGGCGCAACGTTCGCGCTC-AGAGGCGATAATCCACAGGGGA; SEQ ID NO:12) andRGD21-B (CGCGTCCCCTGT-GGATTATCGCCTCTGAGCGCGAACGTTGCGCCTTGAGC; SEQ IDNO:13). The RGD₂₁ envelope proteins were cloned into an expressionplasmid that contained a Zeocin™ selection marker (Invitrogen, Carlsbad,Calif.). RGE₂₁ was constructed using methods analogous to those used forRGD₂₁. Chimeric envelope proteins expressing two RGD sequences, RGD₂₁-15and RGD₂₁-16, were constructed by removal of the Bst EII/Cla I fragmentof RGD₂₁-1, and insertion of the Bst Eli/Cla I region from RGD₂₁-4 andRGD₂₁-9, respectively. TABLE 1 Description of RGD viruses. Position ofLigand Insertion (A.A. Deletion of ENV # Location) #of InsertsNucleotides in Env. RGD ₁₃[CAAA-GRGDSP-TRC] (SEQ ID NO:8) 1 1 1X 2 1 2X3 1 4X 4 38 1X 5 38 3X 6 38 1X 5990-6082 7 68 1X 8 68 2X 9 68 1X6082-6191 10 120 1X 11 120 2X 6238-6281 12 120 3X 13 185 1X 14 230 1X 15230 2X 16 235 1X 17 235 4X 18 310 1X 19 310 2X 20 321 1X 21 321 2X 22382 1X 23 382 2X 24 382 3X 25 388 1X 26 388 2X RGD ₂₁[CAAAQGATFALRGDNPQG-TRC] (SEQ ID NO:11) 1 1 1X 2 38 1X 3 38 1X 5990-6082 4 681X 5 68 1X 6082-6191 6 120 1X R 120 1X 6238-6281 8 185 1X 9 230 1X 10235 1X 11 310 1X 12 321 1x 13 382 1X 14 388 1X 15 1,68 1X,1X 16 1,2301X,1x RGE ₂₁[CAAA-QGATFALRGENPQG-TRC] (SEQ ID NO:25) 1 1 1X 2 38 1X5990-6082 3 68 1X 4 68 1X 6082-1916 5 230 1X

The core of the RGD₁₃ ligand is a six amino acid peptide, GRGDSP (SEQ IDNO:14), which represents an RGD consensus sequence. The core of theRGD₂₁ ligand is a 14 amino acid sequence, QGATFALRGDNPQG (SEQ ID NO:15),derived from the mouse laminin protein (Aumailley et al., 1990, FEBSLett. 262:82-86). Both the RGD₁₃ and RGD₂₁ peptides were flanked bycysteine residues to constrain the sequence within a loop (Aumailley etal., 1990, supra; Yamada et al., 1993, J. Biol. Chem. 268:10588-10592;Hart et al., 1994, J. Biol. Chem. 269:12468-12474; Pierschbacher andRuoslahti, 1987, J. Biol. Chem. 262:17294-17298).

In some cases, chimeric envelope proteins with multiple ligands intandem were also generated. Several of the chimeric envelope proteinshad deletions of envelope sequences, in addition to ligand insertions,as a result of multiple restriction enzyme cleavages. In total, 26chimeric envelope proteins containing the RGD₁₃ ligand, 16 chimericenvelope proteins containing the RGD₂₁ ligand, and five chimericenvelope proteins containing an RGE₂₁ ligand, a control non-bindingpeptide (Aumailley et al., 1990, supra; Hart et al., 1994, supra;Solowska et al., 1989, J. Cell Biol. 109:853-861; Greenspoon et al.,1993, Biochemistry 32:1001-1008), were constructed.

The information provided in this Example provides guidance forconstruction of chimeric envelope proteins containing heterologous shortpeptide ligands.

Example 3 Transduction of Cells with Viruses Containing ChimericEnvelope Proteins

Pseudotyped viruses were generated that express the chimeric envelopeproteins as were control viruses that expressed wild-type ecotropicenvelope protein and that expressed the envelope protein from anamphotropic virus. None of the pseudotyped viruses contained a wild typeenvelope gene. This feature provides an advantage for altering viraltropism since all of the envelope genes in the pseudotyped virus willcontain the heterologous short peptide ligand, thus providing more sitesfor binding to the target host cell.

Plasmids used to express control ecotropic virus, ECO (wild type), weregenerated by expressing the wild-type ecotropic envelope gene encoded bythe plasmid pCEE. Another control was an amphotropic virus, AMPH, whichcontains an amphotrophic viral envelope protein. This virus wasgenerated by expressing the amphotropic envelope, encoded by theexpression vector pCAA The pCAA expression vector was generated byremoving the amphotropic envelope gene from a full-length infectiousclone (Ott et al., 1990, J. Virol. 64:757-766) and engineering it intothe expression vector.

A packaging construct for use in the experiments, LAPNL, was generatedby removal of the VSV-G envelope from LGRNL (Yee et al., 1994, MethodsCell Biol. 43:99-112) and insertion of the secreted alkaline phosphatasegene (SEAP) into LGRNL, producing the packaging construct LAPNL.Transfection with this packaging construct was measured by assaying forthe secreted alkaline phosphatase. The SEAP assay was performed asdescribed by Tropix, Inc. and measured in a luminometer (Moonlight 2010,Analytical Luminescence Laboratory).

Pseudotyped virus containing chimeric envelope proteins was generatedusing a human 293T cell-based packaging cell line, Anjou 65 (Pear etal., 1993, Proc. Natl. Acad. Sci. USA 90:8392-8396). The pseudotypedvirus producer cell lines were generated by cotransfection of Anjou 65cells with LAPNL and a plasmid expressing a chimeric envelope proteinusing Dotap (Boehringer) followed by selection in Zeocin™ (200 μg/ml)for two weeks. RGD₁₃ required cotranfection with a Zeocin expressionplasmid (Invitrogen, Carlsbad, Calif.).

Pseudotyped virus was harvested from viral producer cell lines. Virionassociated reverse transcriptase (RT) activity was performed aspreviously described to measure RT activity of harvested viralsupernatant. RT/PCR was performed by first generating cDNA from 5 μl ofharvested pseudotyped virus using the protocol for Superscript II (GibcoBRL) and oligo-dT with 0.1% NP40. Oligonucleotides to the neomycin genein the LAPNL packaging vector were used to generate the PCR product fromthe cDNA. These oligonucleotides were labeled N1(TTTTGTCAAGACCGACCTGTCC; SEQ ID NO:16) and N2 (CGGGAGCGGCGATACCGTAAAG;SEQ ID NO:17). Target cells were infected as described in Kasahara etal. (1994, Science 266:1373-1376) and Cosset et al. (1995, J. Virol.69:6314-6322). Briefly, 24 hours before infection, NIH 3T3 and A375human melanoma cells were seeded on 60 mm plates at 2×10⁵ cells/plate.Infected cells were seeded onto 150 mm plates and selected for two weekswith 1.0 mg/ml of G418. Colonies were fixed and stained with Giemsa asdescribed in Russell et al. (1993, Nucleic Acids Res. 21:1081-1085).Human fibrosarcoma HT 1080 cells and canine kidney cells MDCK were alsoinfected, examined, and selected in 600 μg/ml of G418. Transductionefficiency was determined by SEAP measurements and by counting coloniesusing a BioRad digital camera and scanner.

Immunoblotting of purified virions indicated that, in all cases tested,the chimeric envelope proteins were incorporated into the virion andcorrectly processed. The viruses expressing the chimeric envelopeprotein with short RGD peptide ligand (RGD viruses) were initiallytested for their ability to transduce mouse NIH 3T3 cells. Data from themouse cell transduction experiments are shown in FIGS. 1 and 2. Thesedata show that many of the RGD viruses retained their ability totransduce mouse cells but those bearing insertions within the N-terminus(RGD₁₃-4,5; RGD₂₁-2,3), VRA (RGD₁₃-8,12; RGD₂₁-5) and C-terminal region(RGD₁₃-19,23,34; RGD₂₁-15,16) did not. Several of these latter RGDviruses also failed to transduce human cells (RGD₁₃-12,19,23,24;RGD₂₁-5,15,16), whereas for others (RGD₁₃-4,5,8; RGD₂₁-2,3) the defectwas mouse cell specific. In addition, most RGD₂₁ viruses transduced NIH3T3 cells with comparable efficiencies to the equivalent RGD₁₃ viruses,and none of the RGD₂₁ viruses transduced NIH 3T3 cells with greaterefficiency than the equivalent RGD₁₃ virus.

Thus, chimeric envelope protein containing a heterologous short peptideligand, when expressed in a packaging system can effectively infect acell from an organism other than the natural host of the parent virus,thus the host range of the virus can be altered by creating viruses withheterologous short peptide ligands in their envelope protein.

Example 4 Transduction of Cells Expressing Integrin Receptors

To further assess the ability of RGD viruses to infect non-mouse cells,the viruses were tested for their ability to transduce A375 humanmelanoma cells. A375 cells have been used to study integrin receptorbinding (Gehlsen et al., 1992, Clin. Exp. Metastasis 10:111-120; Pfaffet al., 1993, Exp Cell Res. 206:167-176; Allman et al., 2000, Eur. J.Cancer 36:410-422). As expected, viruses containing unmodified MLVenvelope failed to transduce this human cell line. Significantly,however, many of the RGD viruses were able to transduce A375 humanmelanoma cells (FIGS. 1 and 2). Transduction occurred when the RGDpeptide was inserted at the N-terminus (RGD₁₃ 1-3; RGD₂₁-1), within theN-terminal region (RGD₁₃ 4-6; RGD₂₁-2,3), within the VRA region(RGD₁₃-7,8,10,11; RGD₂₁-4,6,7), and upstream of the PRR (RGD₁₃-14,15;RGD₂₁-8,9).

RGD viruses with insertions in the PRR (proline-rich region) andC-terminal region failed to transduce human cells. Thus, in constructingchimeric envelope proteins, the PRR is generally not a preferred sitefor insertion of a heterologous short peptide ligand. Several of the RGDviruses that transduced human cells, failed to transduce NIH 3T3 cells(RGD₁₃-4,5,8 and RGD₂₁-2), indicating that viral tropism can beeliminated for the natural host and altered to target a different host.

In all cases tested, RGD viruses that transduced A375 human melanomacells also transduced other human and non-human cell lines thatcontained integrin receptors. This shows that the host range for a viruscan be greatly changed and expanded by introducing a chimeric envelopeprotein containing a heterologous short peptide ligand. Furthermore,these viruses can be targeted to infect a specific host cell.

Example 5 Specificity of Transduction by Virus Containing ChimericEnvelope

To examine the basis and specificity of human cell transduction, twoexperimental approaches were undertaken. In the first approach, theRGD₂₁ ligand was replaced with the corresponding RGE₂₁ sequence.Pseudotyped virus expressing an RGE₂₁ chimeric envelope derivativetransduced NIH 3T3 host cells with efficiencies comparable to theequivalent RGD₂, derivative. However, transduction of A375 humanmelanoma cells was significantly reduced (FIG. 3).

In a second approach, the effect on transduction with RGD viruses wasexamined in the presence of antibodies that bind integrin receptors. Inthese experiments, NIH 3T3 and A375 human melanoma cells were pretreatedwith integrin receptor antibodies, and transduction was performed withtwo of the RGD₂₁ viruses. Briefly NIH 3T3 and A375 human melanoma cellswere pretreated with polyclonal antibodies to β₁, β₃, and α_(v) integrinreceptors (Santa Cruz Biotechnology). For pretreatment, the threeantibodies were diluted 1:100 in DMEM medium and incubated with thecells for four hours. Cells were then incubated with pseudotyped virus(RGD₂₁-1, RGD₂₁-4, or RGD₂₁-9) for six hours. The infected cells werethen analyzed for transduction as described above (see FIG. 1). It wasobserved that transduction of human but not mouse cells wassubstantially reduced (FIG. 4).

Example 6 Chimeric Envelope Proteins Containing GRP Heterologous ShortPeptide Ligands

To test the applicability of the invention to heterologous short peptideligands in addition to integrin ligands, heterologous short peptideligands from bombesin (GRP) and heregulin (HRG) were identified andcloned into MLV ecotropic envelope using methods known in the art.

The sequence of the GRP and HRG ligands are shown in Table 2. A 21 aminoacid GRP sequence, containing 14 residues of the bombesin protein, wasinserted at various locations within the MLV ecotropic envelope togenerate 14 GRP chimeric envelope proteins (Table 2). For the chimericenvelope proteins, GRP 1-14 and HRG 1-9, the position of ligandinsertion and any additional modifications are indicated. GRP chimericenvelope proteins (GRP 1-14) were generated by inserting the 21 aminoacid GRP ligand into the Mlu I and Not 1 sites of previously constructedchimeric envelopes. The sequence encoding CAAAEQRLGNQWAVGHLMTRC SEQ IDNO:18) was generated by annealing two oligonucleotides: GRP_(A)GGCCGAGCAGCGCCTGGGCAACCAGTGGGCCGTCGGCCACCTGATGA; SEQ ID NO:19) andGRP_(B) (CGCGTCATCAGGTGGCCGACGGCCCACTGGTTGCCCAGGCGCTGCTC; SEQ ID NO:20).HRG chimeric envelope proteins (HRG 1-9) were generated by inserting amodified 49 amino acid binding region of the heregulin-β protein(Ballinger et al., 1998, J. Biol. Chem. 273:11675-11684) into the Mlu Iand Not I sites of previously constructed chimeric envelopes. The 49amino acid HRG sequence was derived by annealing four oligonucleotides:HRG_(A)(GGCCGCTTCACACCTTGTAAAGTGCGCAGAGAAGGAAAAGACGTTCTGC-GTCAACGGCGTGAGTGTTACAG;SEQ ID NO:21), HRG_(B)(GCCGTAGGTCTTAAC-CCTGTAACACTCACCGCCGTTGACGCAGAACGTCTTTTCCTTCTCTGCGCACTTTACAAGGTGTGAAGC; SEQ ID NO:22), HRG_(C)(GGTTAAGACCTACGGCTATCTGATGTGCA-AGTGTCCGAACGAGTTCACGGGTGACCGGTGCCAGAACTACGTCATCGTCGA; SEQ ID NO:23), and HRGD(CGCGTCGACGCGATGACGTAGTTCTGGCACCGGTC-ACCCGTGAACTCGTTCGGACACTTGCACATCAGATA;SEQ ID NO:24). Experiments using the HRG chimeric envelope proteins arediscussed below (Example 8). TABLE 2 Description of GRP and HRG virusesPosition of Ligand Insertion Deletion of (A.A. Nucleotides ENV #Location) in Envelope GRP CAAA-EQRLGNQWAVGHLM-TRC (SEQ ID NO:18) GRP-1 1GRP-2 38 GRP-3 38 5990-6082 GRP-4 68 GRP-5 68 6082-1916 GRP-6 120 GRP-7120 6238-6281 GRP-8 185 GRP-9 230 GRP-10 235 GRP-11 310 GRP-12 321GRP-13 382 GRP-14 388 Del. 3 A.A. | FM DPSRYL M HRG CAAA- (SEQ ID NO:26)SHLVKCAEKEKTFCVNGGECYRVKTYGYLMCKCP NEFTGDRCQNYVIAS-TRC HRG-1 1 HRG-2 38HRG-3 38 5990-6082 HRG-4 68 HRG-5 68 6082-1916 HRG-6 120 HRG-7 185 HRG-8230 HRG-9 235

Pseudotyped virus producer cells were generated for each chimericenvelope derivative and the resultant GRP viruses were initially testedfor transduction of host NIH 3T3 cells. Briefly, NIH 3T3 cells, humanA375 melanoma cells, and human MDA-MB-231 breast carcinoma cells wereinfected with a GRP virus, selected with G418 for two weeks, fixed,stained with Giemsa and colonies counted. Amphotropic (Amph) andecotropic viruses (Eco) were generated by expressing the wild typeamphotropic and ecotropic envelopes, pCAA and pCEE, respectively. Theamphotropic envelope, pCAA, and the LAPNL packaging vectors weregenerated as described herein and as is practiced in the art; the latterexpresses the secreted alkaline phosphatase gene (SEAP) and the neomycinresistance gene. FIG. 4 (note the log scale) shows that all of the GRPviruses transduced NIH 3T3 cells except when the ligand was insertedwithin the N-terminal region (GRP-2, GRP-3) or in one case within theVRA (GRP-5). In general, the GRP viruses transduced NIH 3T3 cells withefficiencies comparable to that observed for RGD viruses.

A375 human melanoma and 231 breast carcinoma cells overexpress the GRPreceptor (Yano et al., 1992, Cancer Res. 52:4545-4547; Pansky et al.,1997, Eur. J. Clin. Invest. 27:69-76; Miyazaki et al., 1998, Eur. J.Cancer 34:710-717). GRP viruses with insertions at the N-terminus(GRP-1), within the N-terminal region (GRP-2, GRP-3), within the VRA(GRP-4, GRP-5), downstream of the VRB (GRP-8) and upstream of the PRR(GRP-9) transduced both of these human cell lines. In contrast, GRPviruses with insertions within the PRR (GRP-10) or C-terminal region(GRP-11-GRP-14) failed to transduce human cells.

Example 7 Requirement for GRP Receptor Expression

Experiments were performed to confirm that expression of the GRPreceptor is required for GRP viruses to transduce human cells. First, itwas tested whether treatment of GRP viruses with an antibody to the GRPprotein would block transduction of human cells. GRP-1 or GRP-2 viruseswere pretreated with 2A11 antibody (provided by Dr. Frank Cuttitta). NIH3T3, A375 human melanoma cells, or MDA-MB-231 breast carcinoma cellswere then infected with 2A11 antibody treated GRP or untreated virus andtransduction analyzed as described above (see FIG. 4). The 2A11 antibodywas added to pseudotyped virus at a 1:100 dilution followed byincubation at 4° C. for four hours and then viral infection wasanalyzed. FIG. 6A shows that 2A11, an antibody to the C-terminal regionof GRP protein, substantially reduced transduction of both human cancercell lines but not mouse NIH 3T3 cells. Thus, GRP is required fortransduction of human but not mouse cells.

The question of whether expression of the GRP receptor is required fortransduction of human cells by GRP viruses was examined. Human 293 cellsdo not express the GRP receptor (Valdenaire et al., 1998, FEBS Lett.424:193-196). A 293 cell line was developed that constitutivelyexpresses the GRP receptor (293-GRPR cells) using methods known in theart. Briefly, the GRPR-Zeo construct was generated by insertion of theGRP receptor gene (GRP—R) (provided by Dr. James F. Battey, NIH) intopcDNA3.1/Zeo+ (Invitrogen). 293-GRPR-Zeo cells were generated bytransfection of 293 kidney cells with GRPR-Zeo, selection with Zeocin™,and verification of GRP receptor expression by RT/PCR. 293-GRPR-Zeocells were infected with the GRP-1 or GRP-4 virus, with or withoutpreincubation with the 2A11 antibody and transduction analyzed asdescribed herein. FIGS. 6A and 6B show that 293-GRPR cells, but not theparental 293 cells, were transduced by GRP viruses and that pretreatmentwith the 2A11 antibody blocked transduction.

In a similar experiment, the requirement of the GRP receptor fortransduction of mouse cells by GRP-2, GRP-3 and GRP-5 viruses wasinvestigated. In these experiments, NIH 3T3 and Swiss 3T3 cells wereinfected with a GRP virus and transduction analyzed as described herein.FIG. 6C shows the results of these experiments. Several of the GRPviruses transduced mouse Swiss 3T3 cells, which express the GRPreceptor, but not NIH 3T3 cells, which lack the GRP receptor.Collectively, the results shown in FIGS. 5 and 6 indicate thattransduction of human cells by GRP viruses requires a virus bearing achimeric GRP envelope derivative and a cell expressing a GRP receptor.

Example 8 Chimeric Envelope Proteins Containing HRG Heterologous ShortPeptide Ligands

To test the ability of another heterologous short peptide ligand toalter retroviral tropism when inserted into an envelope protein, aseries of chimeric envelope proteins containing the 56 amino acidheregulin-β peptide sequence (HRG; Table 2) were constructed. Apolypeptide of residues 177 to 226 of HRG binds to and activates theHER3 and HER4 receptor, and was selected as the target ligand (Barbacciet al., 1995, J. Biol. Chem. 270:9585-9589). This ligand was modifiedthrough eleven substitutions known to increase its affinity for thehomodimeric HER3 (Ballinger et al., 1998, J. Biol. Chem.273:11675-11684; Table 2). Sequence encoding HRG ligand was insertedinto MLV envelope gene locations that resulted in chimeric envelopeproteins that had enabled transduction of human cells by GRP viruses andRGD viruses supra.

HRG viruses were first tested for their ability to transduce NIH 3T3cells then MDA-MB-453 and MDA-MB-231 breast carcinoma cells wereinfected with an HRG virus and transduction analyzed as describedherein. The transduction efficiencies of the HRG-8 and HRG-9 viruseswere comparable to the equivalent GRP viruses (see GRP-9 and GRP-10;FIG. 4). By contrast, the transduction efficiencies of the HRG-1, HRG-4,HRG-6 and HRG-7 viruses were significantly lower than the equivalent GRPviruses (GRP-1, GRP-4, GRP-6, GRP-8).

MDA-MB-453 breast carcinoma cells overexpress EGFR family members,whereas MDA-MB-231 breast carcinoma cells do not (Baulida and Carpenter,1997, Exp. Cell Res. 232:167-172; Jeschke et al., 1995, Int. J. Cancer60:730-739; Chan et al., 1995, J. Biol. Chem. 270:22608-22613). FIG. 7Ashows that the HRG-1 and HRG-8 viruses transduced MDA-MB-453 but notMDA-MB-231 cells. The HRG-1 and HRG-8 viruses also transduced two otherhuman breast cancer cell lines that overexpress EGFR family members:MCF-7 and AU-565 cells. In contrast, HRG-2, HRG-3, HRG-4, HRG-5, andHRG-7 failed to transduce MDA-MB-453 cells. This differs from theresults with chimeric envelope proteins that have insertions of GRPpeptide ligands in corresponding loci.

Experiments were conducted to test whether antibodies to HER-3 and HER-4receptors block transduction of human cells by HRG viruses. In theseexperiments, NIH 3T3 and MDA-MB-453 breast carcinoma cells werepretreated with antibodies to HER-3 and HER-4 receptors (Lab VisionCorporation) and then infected with the HRG-1 or HRG-8 virus.Transduction was analyzed as described herein. Pretreatment ofMDA-MB-453 cells with HER3 and HER4 antibodies substantially decreasedtransduction by HRG-1 and HRG-8 viruses indicating that viral entry wasmediated by the HRG-receptor interaction (FIG. 7B).

Example 9 Use of Pseudotyped Viruses with Chimeric Envelope Proteins forKilling Cancer Cells

One use for viruses containing chimeric envelope proteins that redirecthost specificity is for delivery of therapeutically useful genes totarget cells such as cancer cells. Experiments were performed to testwhether retroviruses bearing an appropriate chimeric envelope derivativecan deliver a therapeutically useful gene to cancer cells. Mammaliancells expressing the herpes simplex virus thymidine kinase (TK) gene arekilled by treatment with ganciclovir (Cheng et al., 1983, Proc. Natl.Acad. Sci. USA 80:2767-2770). The GRP-1 virus carrying the HSV TK genewas used to transduce A375 human melanoma and MDA-MB-231 breastcarcinoma cells.

Briefly, A375 human melanoma cells and MDA-MD-231 human breast carcinomacells were infected with GRP-1 virus expressing either the SEAP or TKgene. The packaging vector, LTKNL, containing the TK gene, was generatedby removal of the SEAP gene from an LAPNL packaging vector and insertionof the thymidine kinase gene (TK; provided by Steve Jones, University ofMassachusetts Medical School). GRP virus with the LTKNL packagingconstruct was generated and used to transduce human cells. Cells wereselected with G418 for two weeks, followed by isolation of colonies andculture in media containing 10 μg/ml ganciclovir (Moravek Biochemicals,Inc.) and the cell densities were examined using a Zeiss Axiophotmicroscope.

Following ganciclovir treatment of transduced melanoma and breastcarcinoma cells significant cell death was evident, whereas there was nocytopathic effect in ganciclovir treated cells transduced by a controlGRP-1 virus not expressing the TK gene.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A chimeric retrovirus envelope protein comprising an ecotropicenvelope protein and a heterologous short peptide ligand inserted withinthe ecotropic envelope protein.
 2. The chimeric envelope protein ofclaim 1, wherein the ecotropic envelope protein is a Murine LeukemiaVirus (MLV) envelope protein.
 3. The chimeric envelope protein of claim1, wherein the ecotropic envelope protein is a wild type envelopeprotein.
 4. The chimeric envelope protein of claim 1, wherein theheterologous short peptide ligand is selected from the group consistingof an RGD ligand, a human epidermal growth factor receptor (HRG) ligand,or a gastrin releasing protein (GRP) ligand.
 5. The chimeric envelopeprotein of claim 1, wherein the heterologous short peptide ligand isflanked by at least one cysteine on each side.
 6. The chimeric envelopeprotein of claim 1, wherein the heterologous short peptide ligand isinserted into a conserved region of a wild-type envelope protein.
 7. Anucleic acid molecule comprising a nucleic acid sequence encoding therecombinant chimeric envelope protein of claim
 1. 8. A vector comprisinga nucleic acid sequence encoding a chimeric envelope protein thatcontains a heterologous short peptide ligand.
 9. The vector of claim 8,wherein the vector further comprises a nucleic acid sequence thatencodes a therapeutically useful polypeptide.
 10. A recombinantretroviral particle comprising a chimeric envelope protein comprising aheterologous short peptide ligand.
 11. The recombinant retroviralparticle of claim 10, wherein the retroviral particle can infect a mousecell.
 12. The recombinant retroviral particle of claim 10, wherein theretroviral particle cannot infect a mouse cell.
 13. A method of alteringretroviral tropism, the method comprising (a) introducing into thegenome of a retrovirus a nucleic acid sequence that encodes a chimericenvelope protein, and wherein (b) the nucleic acid sequence of thechimeric envelope protein comprises a heterologous short peptide ligand,thereby producing a pseudovirus having altered tropism.
 14. The methodof claim 13, wherein murine leukemia virus (MLV) retroviral tropism isaltered.
 15. The method of claim 13, wherein the pseudovirus does notexpress wild-type envelope protein.
 16. The method of claim 14, whereinthe heterologous short peptide ligand is inserted into a conservedregion of a wild-type envelope protein.
 17. A method of identifying anucleic acid sequence encoding a chimeric envelope protein that altersviral tropism, the method comprising (a) introducing into the genome ofa retrovirus, a nucleic acid sequence encoding a recombinant envelopeprotein comprising a heterologous short peptide ligand to produce arecombinant virus; (b) infecting a target host cell with the virus; and(c) assaying transduction of the target host cell by the virus, suchthat transduction of the host cell by the virus indicates that thenucleic acid sequence encodes a chimeric envelope protein that altersviral tropism.
 18. The method of claim 17, wherein the virus is an MLV.19. The method of claim 17, wherein the heterologous short peptideligand is in a conserved region of the MLV envelope protein.
 20. Themethod of claim 17, wherein the target host cell is a human cell. 21.The method of claim 17, wherein the target host cell is a cancer cell.22. The method of claim 17, wherein the target host cell comprises amutant gene and the retrovirus comprises a wild type nucleic acidsequence corresponding to the mutant gene.
 23. The method of claim 17,wherein the chimeric envelope protein contains an RGD ligand, an HRGligand, or a GRP ligand.
 24. A method of delivering a nucleic acidsequence to a cell, the method comprising, (a) providing a cell; and (b)infecting a cell with a virus comprising a chimeric envelope protein andthe nucleic acid sequence, wherein the chimeric envelope proteincomprises a heterologous short peptide ligand.
 25. The method of claim24, wherein the heterologous short peptide ligand is an RGD ligand, anHRG ligand, or a GRP ligand.
 26. The method of claim 24, wherein thecell is a mammalian cell.
 27. The method of claim 24, wherein the cellis a human cell.
 28. The method of claim 24, wherein the cell is acancer cell.
 29. The method of claim 24, wherein the cell is in ananimal.
 30. A method of treating cancer, the method comprising (a)providing a cancer cell; and (b) infecting a cancer cell with a virus,the virus comprising a chimeric envelope protein comprising aheterologous short peptide ligand and a therapeutically useful gene. 31.The method of claim 30, wherein the virus is a retrovirus.
 32. Themethod of claim 30, wherein the cancer is in a mammal.
 33. The method ofclaim 30, wherein the cancer is in a human.
 34. The method of claim 30,wherein the therapeutically useful gene is encodes thymidine kinase.